Aggregation is a well known problem in the production of (bio-) pharmaceuticals, especially proteins such as monoclonal antibodies and viruses. More background on protein aggregation may be found in the following review (Wei Wang, 2005). Aggregation can be associated with high production/manufacturing losses, instability, reduced shelf life, adverse effects upon administration, different immunogenic reactions, and up to disease formation itself (like e.g., Alzheimer Parkinson (Taylor et al. 2002), prion encephalopathy and Huntington's). Aggregation may be prevented by careful selection of buffers or media applied in the production process (Gu et al., 2003, Cromwell et al. 2006). Addition of compounds like urea or guanidinium salts has been known to solubilize proteins. However, these agents also affect protein structure and efficacy. Suppressing or preventing aggregation is not always successful and compromises have to be made which may lead to (relatively) high product losses during manufacturing, storage or loss of efficacy over time.
During the production of viral components such as e.g. Sabin based inactivated poliovirus, unwanted aggregation is also known to occur, which may give rise to large variations in purification product recovery (yield) in viral (components) manufacturing. There is thus a need for an improved method for the production of viral components.
Viruses are infectious agents that can only replicate inside living cells, depending on the virus they may infect different types of organisms such as animals, plants, bacteria and archaea (Koonin E V et al. 2006). The virus consists of two or three distinct parts, the genetic material and a viral protein coat, sometimes supplemented by a lipid-bilayer membrane or envelope. The viral coat is made up out of multiple proteins, which form a highly complex quaternary structure in a helical, isocahedral or even more complex structure. More background on viruses may be found in the following reference (Fields Virology, 2007).
In our study we focused on poliovirus and influenza as a model for non-enveloped and enveloped viruses, respectively. Poliovirus is commonly used as a non-specific model virus for non-enveloped RNA viruses in viral removal validation studies as representative of Picornaviruses in general (technical note Millipore: AN1650EN00, www.bioreliance.com/library/?id=90, http://www.criver.com/files/pdfs/bps/bp_r_viral_tse-clearance_studies.aspx). Furthermore poliovirus is a well studied virus on which a huge body of scientific literature is available making it a suitable candidate. As a representative of enveloped viruses different strains of influenza were used. Influenza is a virus which is causing a great deal of concern each year due to it's variability (antigenic drift). Influenza is studied worldwide and due to the disease burden a likely target for any vaccine improvement. The high variability make influenza virus a suitable representative to quickly test this method for reducing and preventing of aggregation against many variations, showing the wide range in which this technique may be used.
Poliomyelitis, also referred to as polio or infantile paralysis, is an infectious viral disease caused by three related virus serotypes: poliovirus type 1, 2 and 3. Polioviruses belong to the genus Enteroviruses in the Picornaviridae family. In humans, polioviruses are mainly acquired by fecal-oral or oral-oral transmission. After infection, poliovirus proliferates in the gastrointestinal tract, and from there it can enter the central nervous system. Such an infection may cause paralysis. Polio cannot be cured. However, it can be prevented by vaccination. Currently, there are two safe and effective polio vaccines available in the market: Oral Polio Vaccine (OPV), and Inactivated Polio Vaccine (IPV). OPV is based on life-attenuated strains of the poliovirus (the so-called Sabin strains, after Albert Sabin, who first developed OPV), this vaccine is administered via the oral route. In contrast, IPV, is based on using purified wild-type poliovirus strains, which are chemically killed and is administered intramuscular by injection. IPV was first developed by Jonas Salk [there are several reviews available on polio and polio vaccines: Koch and Koch, 1985; Duchene et al., 1990; Kew et al. 2005; Heinsbroek and Ruitenberg, 2010].
Both available polio vaccines (OPV and IPV) provide high levels of protection from paralytic poliomyelitis. OPV has thus far been the vaccine of choice for the global polio eradication as it has several advantages: easy to administer and less expensive. However, in some cases OPV may cause vaccine associated paralytic poliomyelitis (VAPP) or may lead to vaccine derived poliovirus (VDPV), and should be preferably discontinued as soon as eradication is successful [Kew et al., 2005; Heymann et al., 2005 & 2006; Chumakov et al., 2007; Nathanson & Kew, 2010; Aylward and Tangermann, 2011]. It is also not excluded that OPV might revert back to the wild type variant (Lee et al 2012). Therefore, the need for new, safe and effective polio vaccines is increasing. The pathway to a global post-eradication polio vaccination policy depends on, amongst others, the availability and price of IPV [Heinsbroek and Ruitenberg, 2010; Thompson and Tebbens, 2012].
To discontinue the use of OPV after polio eradication, and to reduce the cost of IPV per dose, different approaches are being followed. Amongst others, these approaches include: a) IPV based on the attenuated Sabin poliovirus strains (Sabin-IPV) [Bakker et al., 2011; Hamidi & Bakker, 2012]; b) IPV based on newly designed alternative poliovirus seed strains [Chumakov et al., 2008; Robinson H L 2008; Hamidi & Bakker, 2012]; c) IPV produced from alternative mammalian cells that efficiently support poliovirus replication [Hamidi & Bakker, 2012; Sanders et al., 2012; Crucell, U.S. Pat. No. 0,027,317, 2011]. In all such developments, opportunities in cost price reduction can be realized by implementation of known methods in up-stream and down-stream process optimization (e.g. more efficient use of bioreactor capacity), and overall modernization (e.g. using animal-component-free cell and virus culture media, disposable filters and alike).
Currently, IPV is most commonly based on using three wild-type virulent strains, Mahoney (type 1 poliovirus), MEF-1 (type 2 poliovirus), and Saukett (type 3 poliovirus). The polioviruses are grown separately in mammalian cell culture. Subsequently, after several purification steps, the poliovirus is inactivated using formalin (formaldehyde) whereafter they can be mixed to the final desired formulation and filled.
Influenza virus causes an acute respiratory infection, with considerable morbidity and mortality. Prevalence is highest in school-aged children. Small children, elderly and those with conditions such as lung and heart disease, diabetes or severe asthma are at risk for severe influenza. Clinically, influenza comprises acute febrile illness with myalgia, headache and cough.
Although the disease influenza has been known for centuries, the causative agent was long unknown. The first human influenza virus was isolated in 1933.
The virus could be propagated on embryonated eggs (still a common practice, later complemented with the ability to grow the virus on cell cultures), which greatly facilitated the ability to study the virus.
The influenza virus is an RNA viruses of the family Orthomyxoviridae and is composed of a lipid envelope around eight segments of RNA. On the envelope two major proteins (antigens) are present: the neuraminidase (NA/N) and the haemagglutinin (HA/H). Haemagglutinin is the protein that attaches the virus to cells of the respiratory epithelium and subsequently fuses the viral membrane with the membrane of the epithelial cell, to allow the virus entry. The neuraminidase is a viral enzyme that facilitates the release of newly produced viral particles from infected cells.
Besides man, influenza viruses can infect a wide range of animal species, the most relevant for humans being birds and pigs, because the viruses from these species can generally also infect humans. A marked feature of influenza viruses is variability. Variation is driven by immune selection, meaning that the virus will constantly try to escape host immunity. It can do so by gradual mutation of its antigens known as antigenic drift or by swapping entire RNA segments coding for antigens with related strains, known as antigenic shift. Immune selection pertains to antibody responses against the surface antigens, and to a lesser extent to T-cell responses, which are mainly directed against the internal proteins.
Due to antigenic drift, new vaccines against seasonal influenza need to be produced each year, containing the antigens that are expressed on the circulating viral strains in the respective season. Antigenic shift, or a series of antigenic drift mutations, may cause a pandemic. Protection against a pandemic outbreak necessitates the usage of newly developed potent vaccines containing the antigens expressed by the pandemic virus.
As explained herein, the inventors identified an improved method for the production of viral components and for an improved composition comprising such viral components, wherein aggregation is prevented or reduced.